The intrinsic ability of structural protein(s) of many viruses to self-assemble into virus-like particles (VLPs) has led to the development of a specific class of subunit vaccine (Chackerian, 2007; Grgacic and Anderson, 2006; Garcea and Gissmann, 2004). VLPs are generally similar to viruses in shape, size and morphology but are non-infectious due to the lack of infectious component of a virus. VLPs are highly immunogenic and the cellular uptake and the intracellular trafficking of VLPs are similar to viruses (Grgacic and Anderson, 2006). Thus, VLPs combine the advantages of both whole virus as well as subunit vaccines (Chackerian, 2007; Ludwig and Wagner, 2007). VLPs have been expressed using a variety of expression system including bacteria, yeast, transgenic plant, insect and mammalian cell culture systems and against a wide array of viruses infecting human, animals and plants. For example, VLPs made from hepatitis B virus (HBV) major surface antigen are commercially available as Recombivax® (Merck & Co, Inc.) and Energix (GlaxoSmithKline [GSK], Inc.). VLPs assembled from human papillomavirus (HPV) major capsid protein, LI in yeast was approved as a vaccine (Gardasil®, Merck) against cervical cancer in human. A competing product, named Cervarix™ (GSK), made using a baculovirus expression system in insect cell culture was approved in 2006. Both these vaccines elicited a stable antibody response and a long-term protection against infection with cognate virus (Mao et al., 2006; Harper et al., 2006).
VLPs have also been made against many other human viruses such as, Norwalk virus, rotavirus, influenza virus, SARS coronavirus, Ebola virus, and poliovirus (reviewed by Grgacic and Anderson, 2006; Landry et al., 2010). Efforts have also been made to use VLPs as candidate for developing vaccine against animal viruses such as infectious bursal disease of poultry (Remond et al., 2009); canine parvovirus (Gilbert et al., 2004); parvovirus of mice (Hernando et al., 2000); and porcine parvovirus (Sedlik et al, 1999).
In recent years, efforts have been made to develop VLP-based vaccine in shellfish and fish. For example, the capsid protein of infectious hypodermal and hematopoietic necrosis virus, a parvovirus that infects penaeid shrimp (Penaeus sp.), when expressed in Escherichia coli, was found to self-assemble into VLPs that are similar in size and shape to cognate virus and are taken up by the hemocytes in primary culture. This opened up a possibility of using VLPs as vehicles for delivery of antiviral therapies in shrimp (Hou et al., 2009). VLPs have been expressed in mammalian cells (hamster fibroblast cells, BHK-21, McKenna et al., 2001) and in insect larvae (Tricopiusui ni, Shivappa et al., 2005) for an infectious pancreatic necrosis virus (IPNV) that causes infectious pancreatic necrosis in salmonids fish. These VLPs contained both structural proteins, VP2 and VP3, of IPNV, and are similar in size compared to the native virus (McKenna et 5 al., 2001; Shivappa et al., 2005).
More recently, Allnutt and colleagues expressed one of the two capsid proteins, VP2, of IPNV in yeast that self-assembled to sub-viral particles (SVP) (Allnutt et al., 2007). The IPNV SVPs were 22 nm in size compared to the 60 nm size of the VLPs expressed in mammalian cells and in insect larvae. Since most of the antigenic epitopes of IPNV are located in VP2 protein, the yeast expressed IPNV SVP when injected or orally administered into rainbow trout elicited antibody response. In addition, vaccinated fish when challenged with a cognate infectious virus, showed significant reduction in viral load compared to the unvaccinated fish (Allnutt et al., 2007). This showed the potential of using IPNV SVP as a candidate for developing injectable as well as oral vaccine.
Subsequently, to evaluate the potential of IPNV VP2 SVP as a platform to produce bivalent vaccine, a heterologous epitope, human oncogene epitope c-myc, was cloned into IPNV SVP backbone and expressed in yeast. The chimeric VLP when injected into rainbow trout elicited antibody response not only against IPNV but also against the c-myc epitope (Dhar et al., 2010). When fish vaccinated with chimeric VLPs were injected with IPNV, vaccinated fish had a significant reduction in viral load compared to unvaccinated fish. This indicated that IPNV VLP can tolerate the insertion of foreign epitope without affecting the antigenicity of the backbone peptide or the foreign epitope (Dhar et al., 2010). This opened up a possibility of using IPNV VLP as a platform to developing a bivalent vaccine against major viral diseases in salmonids fish.
Infectious pancreatic necrosis (IPN) caused by IPNV and infectious salmon anemia (USA) caused by the infectious salmon anemia virus (ISAV) are the two major viral diseases in salmonid. IPNV is a bi-segmented double-stranded RNA (dsRNA) containing virus belonging to the family Birnaviridae, genus Aquabirnavims. Segment B of the viral genome encodes a RNA-dependent-RNA-polymerase protein, VP1. Segment A encodes a polyprotein which is co-translationally cleaved by the viral-encoded serine-lysine protease, VP4 to make capsid proteins, precursor VP2 (pVP2) and VPS (Duncan et al., 1987; Dobos et al., 1997). Subsequently pVP2 is processed by the host cell protease to form mature VP2 capsid protein (Magyar and Dobos, 1994). VP2 constitutes the outer shell of the viral capsid proteins (Pous et al., 2005). It is quite abundant and contains most of the antibody neutralizing epitopes (Frost et al., 1995; Heppell et al., 1995). Amino acid residues 217 and 221 in the VP2 protein are the major determinants of virulence in IPNV (Santi et al., 2004). VPS, on the other hand, constitutes the inner structural protein and remain bound to viral genomic dsRNA to form the ribonucleoprotein core structure (Pederson et al., 2007). VPS also binds to RdRp protein, VP1. It has been reported that VPS carries some antigenic epitopes. In addition to the capsid protein open reading frame (ORF), Segment A also encodes a small arginine-rich non-structural protein VPS, the biological functions of which is unknown.
Infectious pancreatic necrosis (IPN) is an important disease of finfish worldwide. The disease causes high mortality in fry and post-smolt salmon. In recent years, a number of vaccines against IPN have been commercialized (reviewed by Gomez-Casado et al., 2011). These include Alpha Ject® 1000 (licensed in Norway, Chile and UK, Pharmaq AS, Norway), Bimagen Forte (licensed in Canada, Aqua Health Ltd., Novartis, Canada), IPNV (licensed in Chile, CENTROVET, Chile), Norvax (Intervet-International BV, The Netherlands), and SRS/IPNV/Vibrio (licensed in Canada and Chile, Microtek International Inc., British Columbia, Canada). The first three of these vaccines are composed of inactivated virus while the remaining two contain recombinant VP2. However, the modes of delivery for all of these vaccines are via intraperitoneal injection route. Despite a vaccination program implemented by fish farmers, outbreaks of IPN disease occur from time to time and mortality rates during outbreak vary depending on the genetic susceptibility of the stock, environmental stress and viral strains (Ozaki et al., 2001; Houston et al., 2008; Sundh et al, 2009).
Infectious salmon anemia (USA) was first reported in Atlantic salmon (Salmo salar) from Norway in 1984 (Thorud and Djupvik, 1988). Since then the disease has been reported in Atlantic salmon from Canada, USA and Scotland and Coho salmon (Oncorhynchus kisutch) in Chile (reviewed by Cotte et al., 2010; Gomez-Casado et al., 2011). The cumulative mortality due to ISA outbreak can reach up to 100% (Kibenge et al., 2004). ISAV is an enveloped virus containing eight negative-sense single-stranded linear RNA segments and belongs to the family Orthomyxoviridae, genus Isavirus (Kawaoka et al, 2005). It has been reported that in ISAV the virulence lies mainly in the viral encoded proteins hemagglutinin esterase (HE) and fusion glycoproteins (Kibenge et al, 2007; Muller et al, 2010; Cotte et al, 2010). A number of vaccines have been commercialized against ISA. These include Alpha Jects® and Micro-1 ISA (licensed in Norway, Ireland, Finland and Chile, Pharmaq AS, Norway), FORTE VI (Licensed in Canada, Aqua Health Ltd., Novartis, Canada), ISA vaccine (licensed in Canada, Microtek International, British Columbia, Canada), and ISAV (CENTROVET Ltda., Chile). All but CENTROVET ISAV vaccines are composed of inactivated whole ISAV, and are delivered via injection route. ISAV vaccine of CENTROVET Ltda., on the other hand, contains recombinant HE protein expressed in yeast and is delivered via oral route (Tobar et al., 2010).
As fish farming is expanding globally, vaccinations are playing an increasingly important role in fish health management. An ideal viral vaccine must induce long lasting protection at an early age, prevent carrier formation, and be effective against a large number viral serotypes. Injection cannot be used for small fish; therefore, either oral delivery or immersion are more preferred routes for early vaccination. These attributes of a vaccine must be met either by a recombinant subunit vaccine or by an inactivated viral vaccine, as a live attenuated vaccine could potentially lead to carrier formation.